The invention relates generally to the equilibrative transport of nucleosides into cells, and more particularly to nitrobenzylmercaptopurineriboside (NBMPR)-insensitive, equilibrative, nucleoside transport proteins (iENTPs), to nucleic acids which encode the proteins, methods of use of the proteins and nucleic acids, and antibodies to the proteins.
Aside from being potential precursors to the building blocks of nucleic acids, the natural nucleosides are important metabolites having many physiological effects in assorted organs. For example, adenosine and its corresponding nucleotides are local signaling molecules that act through purinergic receptors to affect such varied physiological functions as lipolysis, neurotransmitter release, coronary vasodilation, cardiac contractility, renal vasoconstriction, and bronchial constriction; and thus extracellular adenosine concentrations can have significant effects on cardiac and vascular functions as well as play a role in neuromodulation [reviewed by Griffith et al. Biochim. Biophys. Acta Rev. Biomembr., 1286:153-181 (1996); Cass, in Drug Transport in Antimicrobial Therapy and Anticancer Therapy (N. H. Georgopapadakou, ed.(Marcel Dekker)), 403-451 (1995)]. In addition, nucleoside analogs are presently employed as anti-retroviral drugs, and as anticancer drugs. Although some extracellular nucleosides can passively permeate the plasma membrane, most participate in some form of protein mediated transport performed by nucleoside transport proteins. Nucleoside transport proteins play an important role in the uptake and efflux of physiological nucleosides used in DNA and RNA synthesis, lipid and glycogen metabolism, and glycoprotein and glycolipid synthesis. Furthermore nucleoside transport proteins mediate the uptake and efflux of a number of antitumor and antiviral nucleoside analogs in cells [Cass, 1995, supra]. Nucleoside transport inhibitors are currently being investigated as modulators of adenosine action in cerebral and cardiac ischemia to provide protection from reperfusion injury [Rongen et al., J. Clin. Invest. 95:658-668 (1995); Parkinson et al., Gen. Pharmacol. 25:1053-1058 (1994)].
The first nucleoside transporters studied functioned as facilitated diffusion systems. Such equilibrative nucleoside transport proteins were initially classified solely by their sensitivity to nitrobenzylmercaptopurineriboside (NBMPR). As the study of these proteins progressed, additional characteristics such as permeant selectivity and tissue distribution have been used to further distinguish these proteins [Griffith and Jarvis, Biochim. Biophys. Acta Rev. Biomembr., 1286:153-181(1996)]. More recently, sodium-dependent concentrative nucleoside transport proteins have also been identified.
At least five distinct nucleoside transport activities have been identified that differ in their permeant selectivity, sensitivity to inhibitors and distribution in normal tissues and tumors [Griffith and Jarvis, 1996, supra]. Two of these activities exhibit equilibrative mechanisms that mediate both the influx and efflux of nucleosides across the plasma membrane, while the other three activities exhibit concentrative, sodium-dependent mechanisms that under physiological conditions mediate only the influx of nucleosides.
The major equilibrative carrier in most cells, es (equilibrative, sensitive) is highly sensitive to the inhibitor NBMPR, having IC50 values of 0.1 to 1 nM. A human homolog of this protein (hENT1) has recently been cloned (Griffiths et al., Nature Med. 3:89-93 (1997). It has 10 to 11 predicted membrane spanning regions and has some structural similarities to the equilibrative glucose carriers. It does not however, share sequence homology with the glucose transporter family and appears to represent a new family of membrane transport proteins designated ENT for equilibrative nucleoside transporter.
Many cells also contain a second equilibrative transporter ei (equilibrative, insensitive) that is insensitive to nanomolar concentrations of NBMPR, but can be inhibited by higher (xcexcM) concentrations [Belt, Mol. Pharmacol., 24:479-484 (1983); Plagemann and Wohlheuter, Biochim. Biophys. Acta, 773:39-52 (1984)]. This protein, an NBMPR-insensitive equilibrative nucleoside transport protein (iENTP) has remained elusive. Both of the equilibrative transporters accept a broad range of physiological nucleosides and their cytotoxic and antiviral analogs as permeants, although there appear to be differences in their affinity for some nucleosides [Griffith and Jarvis, 1996, supra].
iENTPs also are present in most tumor cells, although the level of iENTP appears to be variable. The concentration of iENT in a particular tumor cell is likely to be a major determinant in the ability of that cell to grow following the administration of an es transport inhibitor to block the nucleoside salvage pathway, together with an inhibitor of de novo nucleoside synthesis, such as trimetrexate, methotrexate, and tomudex. The level of iENTP in a tumor cell is also likely to be a determinant of the success of using es inhibitors to block the efflux of cytotoxic and antiviral nucleoside analogs from cells. Under such circumstances, cells with higher concentrations of iENTP will have a higher efflux of cytotoxic and antiviral nucleoside analogs, unless an inhibitor of the iENTP is also administered.
NBMPR and its congeners are the most specific and potent inhibitors of the es transporter currently available. The.es transporter has a high-affinity binding site for NBMPR that overlaps at least in part with the substrate binding site [Jarvis, in Adenosine Receptors, D. M. F. Cooper and C. Londos, eds., (New York: Alan R. Liss, Inc.), pp. 113-123 (1988)]. NBMPR binds to this site with a dissociation constant of 0.1 to 1 nM and completely inhibits nucleoside uptake via es at concentrations in the nanomolar range [Paterson and Cass, in Membrane Transport of Antineoplastic Agents, I. D. Goldman, ed., (New York: Pergamon Press), pp. 309-329 (1986); Gati and Paterson, in The red cell membrane: structure, function, and clinical implications, P. Agre and J. C. Parker, eds., (New York: Marcel Decker), pp. 635-661 (1989); Jarvis, 1988, supra; Plagemann et al., Biochim. Biophys. Acta., 969:1-8 (1988)]. At high concentrations ( greater than 1 uM), however, NBMPR also inhibits the ei transporter [Paterson et al., Mol. Parmacol., 18:40-44 (1980); Belt, Mol. Pharmacol., 24:479-484 (1983); Plagemann and Wohiheuter, Biochim. Biophys. Acta., 773:39-52 (1984)].
Dipyridamole also binds to the NBMPR-binding site of es [Jarvis, Mol. Pharmacol., 30:659-665 (1986)], but is a less potent inhibitor of es than NBMPR [Plagemann and Wohlheuter, Curr. Topics Membr. Trans., 14:225-330 (1980); Paterson and Cass, 1986, supra; Plagemann and Woffedin, Biochim. Biophys. Acta., 969:1-8 (1988)].
Dipyridamole also inhibits the ei transporter, but its potency against this transporter has been unclear. It has been suggested that the es transporter and the ei transporter are equally sensitive to dipyridamole since the curves for inhibition of nucleoside transport are monophasic in cells that possess both transporters [Jarvis, 1988, supra; Plagemann et al., 1988, supra]. However, recent studies with Ehrlich ascites tumor cells in which the es transporter was blocked by addition of low concentrations of NBMPR, suggest that the ei transporter is significantly less sensitive to dipyridamole than es [Hammond, J. Pharmacol. Exp. Ther., 259:799-807 (1991)].
In addition to the two equilibrative nucleoside transporters there are at least three Na+-dependent, concentrative nucleoside transport activities that differ from each other, and from the equilibrative transporters, in their substrate specificity. Two of these, cif and cit (also called N1 and N2), exhibit selectivity for purine and pyrimidine nucleosides respectively [Vijayalakshmi et al., J. Biol. Chem., 263:19419-19423 (1988) and Williams et al., Biochem. J., 264:223-231 (1991)]; while the third, cib (also called N3), has a broader selectivity accepting both purine and pyrimidine nucleosides [Wu et al., J. Biol. Chem., 267:8813-8818 (1992); Huang et al., J. Biol. Chem., 268:20613-20620 (1993)]. All three of the concentrative nucleoside transporters are insensitive to NBMPR and dipyridamole at concentrations up to 10 xcexcM; and under physiological conditions mediate only the influx of nucleoside into cells. These concentrative transport activities have been observed predominantly in normal tissues such as kidney [Le Hir and Dubach et al., Pflugers Arch., 401:58-63 (1984); Williams et al., Biochem. J., 264:223-231 (1989); Williams et al., Biochem. J., 274:27-33 (1991); Le Hir et al., Pflugers Arch., 401:58-63 (1990)] and intestine Schwenk et al., Biochim. Biophys. Acta., 805:370-374 (1984); Vijayalakshtni et al., J. Biol. Chem., 263:19419-19423 (1988); Williams et al., Biochem. J., 274:27-33 (1991), and appear to be the major nucleoside transport activity in the specialized epithelial cells of these tissues [Williams et al., Biochem. J., 274:27-33 (1989); Vijayalakshmi et al., J Biol. Chem., 263:19419-19423 (1988)]. However, low levels of Na+-dependent nucleoside transport have been observed in some tumor cells lines (Lee et al., Biochem. J., 274:85-90 (1991); Belt et al., Mol Pharmacol., 24:479-484 (1993); Crawford et al., J. BioL Chem., 265:13730-13734 (1990b); Dagnino et al., Cancer Res., 50:6549-6553 (1990)].
cDNA clones have recently been obtained for two of the concentrative nucleoside transporters. Cass and co-workers have cloned rCNT1 from rat intestine. This cDNA encodes a 71 Kd protein with cit-type transport activity in transient expression studies in Xenopus oocytes [Huang et al., J. Biol. Chem., 269:17757-17760 (1994)] and COS cells [Fang et al., Biochem. J., 317:457-465 (1996)]. The second transporter, rSPNT (rCNT2) was cloned from rat liver and encodes a 72 Kd protein that has cif-type transport activity in expression studies in Xenopus oocytes. The CNT1 and SPNT transporters are 64% identical in their deduced amino acid sequences, and have significant homology with the bacterial nupC nucleoside transporters. They do not, however, have significant homology with any known mammalian proteins, and thus represent a new family of mammalian membrane transporters. It should be noted that rCNT1 and rSPNT do not share homology with SNST [Pajor et al., J. Biol. Chem., 267:3557-3560 (1992)], a member of the sodium-dependent glucose transporter family that has weak nucleoside transport activity when expressed in Xenopus oocytes. It is not yet known whether SNST represents a significant nucleoside transport activity in mammalian cells. The human homolog of CNTI has recently been cloned [Ritzel et al. Am. J. Physiol. (1997)].
The isolation and cloning of nucleoside transport proteins allows the biochemical characteristics of these transport proteins to be individually investigated and exploited. Such analysis is important for drug development, for example, in which drugs can be more readily designed to inhibit specific transport mechanisms. Unfortunately, heretofore, no NBMPR-insensitive equilibrative transport protein has been isolated or cloned, which has severely hampered analogous studies with this major class of nucleoside transporters.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant invention.
Nucleosides play a central role in cellular metabolism. The nucleoside salvage pathway is an important means employed by cells to maintain the requisite amount of these important metabolites. The initial step in the nucleoside salvage pathway is their transport across the plasma membrane. The key mode of transport of nucleosides into the cell is performed by nucleoside transport proteins contained in the plasma membranes. The present disclosure reports the first isolation and cloning of a cDNA encoding an NBMPR-insensitive equilibrative nucleoside transporter.
The present invention provides a purified transmembrane protein with nucleoside transport activity and the active fragments thereof. The transmembrane protein transports nucleosides across the plasma membrane through a facilitated diffusion process. More specifically, the transmembrane protein is an equilibrative nucleoside transport protein which is insensitive to nitrobenzylmercaptopurineriboside (NBMPR). In one embodiment the NBMPR insensitive, equilibrative nucleoside transport protein (iENTP) contains approximately 450 amino acid residues, and 8 to 12 putative transmembrane domains. In one such embodiment the iENTP is a vertebrate protein. In a preferred embodiment the iENTP is a mammalian protein. In a more preferred embodiment the iENTP is a human protein containing 456 amino acids and has 10 to 11 putative transmembrane domains.
One aspect of the present invention provides an isolated nucleic acid which encodes an iENTP of the present invention that includes exons and introns as shown in FIG. 6. In a preferred embodiment of this type, the isolated nucleic acid contains the nucleotide sequences of SEQ ID NO:5 and SEQ ID NO:10. The introns of the gene are individually part of the present invention,.having nucleotide sequences of SEQ ID NOs:11, 12, 13, 14, 15, 16, 17, 18, and 19 for introns 1-9 respectively. The 5xe2x80x2 portion of intron 10 has the nucleotide sequence of SEQ ID NO:20 whereas the 3xe2x80x2 portion of intron 10 has the nucleotide sequence of SEQ ID NO:21. Intron 11 has the nucleotide sequence of SEQ ID NO:22. Nucleic acid probes which hybridize to the isolated nucleic acid are also included in the present invention. In a preferred embodiment of this type, the nucleic acid probes hybridize to the untranslated portion of the nucleic acid.
The present invention further provides an isolated nucleic acid that contains a nucleotide sequence of the genomic 5xe2x80x2 flanking region of a gene encoding an iENTP. In a preferred embodiment of this type, the isolated nucleic acid has the nucleotide sequence of SEQ ID NO:6. The present invention also includes nucleic acid probes which hybridize to the nucleic acid sequence of SEQ ID NO:6.
Another aspect of the present invention includes isolated nucleic acids encoding the iENTPs and active fragments thereof. One such isolated nucleic acid encodes an amino acid sequence of a transmembrane protein that functions as an equilibrative nucleoside transport protein that is insensitive to NBMPR. In a particular embodiment the nucleic acid encodes an iENTP that contains approximately 450 amino acid residues. In one embodiment of this type, the isolated nucleic acid has a nucleotide sequence with at least 80% similarity with the coding sequence of the human iENTP (hENT2), SEQ ID NO:1. In another embodiment of this type, the isolated nucleic acid has a nucleotide sequence with at least 80% identity with the coding sequence of the human iENTP (hENT2), SEQ ID NO:1. In still another embodiment the isolated nucleic acid has the nucleotide sequence of nucleotides 238-1605 of SEQ ID NO:1. In yet another embodiment of this aspect of the invention, an isolated nucleic acid encodes an iENTP having the amino acid sequence of hENT2, SEQ ID NO:2. In a related embodiment an isolated nucleic acid encodes SEQ ID NO:2 comprising one or more conservative substitutions thereof.
The iENTPs of the present invention, as well as the corresponding nucleic acids which encode them can be obtained from any natural source preferably from a vertebrate cell, more preferably from a mammalian cell, and most preferably from: a human cell.
The present invention also includes oligonucleotides that hybridize to the nucleic acids encoding the iENTIPs of the present invention. In one embodiment the oligonucleotide consists of at least 18 nucleotides. In a preferred embodiment, the oligonucleotide consists of at least 27 nucleotides. In a more preferred embodiment, the oligonucleotide consists of at least 36 nucleotides. Oligonucleotides of the present invention can be used as nucleic acid probes, PCR primers, antisense nucleic acids, and the like, including for diagnostic and therapeutic purposes.
In one such embodiment the oligonucleotide hybridizes to SEQ ID NO:1, or more particularly hybridizes to the coding sequence of SEQ ID NO:1. In a related embodiment the oligonucleotide hybridizes to the nucleotides 512-579 of SEQ ID NO:1. In one embodiment, the hybridization is performed under moderate stringency. In another embodiment, the hybridization is performed under standard hybridization conditions. In yet a third embodiment, the hybridization is performed under stringent hybridization conditions.
Isolated DNAs that encode the iENTPs of the present invention and active fragments thereof are also part of the present invention. In one embodiment, the nucleotide sequence of the DNA has at least 80% similarity with the coding sequence of SEQ ID NO:1. In another embodiment, the nucleotide sequence of the DNA has at least 80% identity with the coding sequence of SEQ ID NO:1. In still another embodiment the DNA has the nucleotide sequence of nucleotides 238-1605 of SEQ ID NO:1. In yet another embodiment the DNA encodes an iENTP having the amino acid sequence of SEQ ID NO:2. In a related embodiment the DNA encodes an amino acid sequence of SEQ ID NO:2 comprising one or more conservative substitutions thereof. In a particular embodiment the DNA is a recombinant DNA (cDNA).
In another embodiment, an isolated or recombinant nucleic acid (including a DNA) has at least 80% similarity with the coding sequence of SEQ ID NO:7. In another embodiment, the nucleotide sequence of the nucleic acid has at least 80% identity with the coding sequence of SEQ ID NO:7. In still another embodiment the nucleic acid contains the nucleotide sequence of SEQ ID NO:7. In yet another embodiment the nucleic acid encodes a protein containing the amino acid sequence of SEQ ID NO:8. In a related embodiment the nucleic acid encodes an amino acid sequence of SEQ ID NO:8 comprising one or more conservative substitutions thereof. In a particular embodiment the DNA is recombinant (cDNA).
All of the isolated nucleic acids and recombinant DNAs of the present invention can further comprise a heterologous nucleotide sequence. Such heterologous nucleotide sequences can encode, for example, a fusion peptide (e.g., a FLAG-tag) or a chimeric protein partner such as a fusion protein.
The present invention also includes DNA constructs comprising the isolated DNAs encoding the iENTPs of the present invention. In one such embodiment the DNA is operatively linked to an expression control sequence. In one embodiment the DNA is operatively linked to an expression control sequence and encodes the amino acid sequence of SEQ ID NO:2. In another embodiment the DNA is operatively linked to an expression control sequence and encodes the amino acid sequence of SEQ ID NO:2 comprising one or more conservative substitutions thereof. In a particular embodiment the DNA is a recombinant DNA (cDNA).
Also included in the present invention are transfected or transduced cells which are transfected or transduced with the recombinant DNA constructs of the present invention. The transfected or transduced cells can be either a prokaryotic cell, or a eukaryotic cell. In one such embodiment, the transfected cell is a COS cell. In another embodiment, the transduced cell is a hematopoietic stem cell. In a particular embodiment, the transfected cell is a human T-cell leukemia CEM cell. In a preferred embodiment the transfected or transduced cell is transfected or transduced with a DNA construct containing a DNA that is operatively linked to an expression control sequence and encodes the amino acid sequence of SEQ ID NO:2. In a related embodiment the transfected or transduced cell is transfected or transduced with a DNA construct containing a DNA that is operatively linked to an expression control sequence and encodes the amino acid sequence of SEQ ID NO:2 comprising one or more conservative substitutions thereof.
Another aspect of the present invention includes the isolated iENTPs of the present invention and active fragments thereof. In its broadest embodiment the isolated iENTP is a transmembrane protein that is NBMPR insensitive, and functions as an equilibrative nucleoside transport protein. In a particular embodiment, the iENTP has approximately 450 amino acids. In one embodiment the iENTP is encoded by a nucleotide sequence having at least 80% similarity with the coding sequence of SEQ ID NO:1. In another embodiment the iENTP is encoded by a nucleotide sequence having at least 80% identity with the coding sequence of SEQ ID NO:1. In still another embodiment the iENTP has an amino acid sequence of SEQ ID NO:2 comprising one or more conservative substitutions thereof. In a preferred embodiment the isolated iENTP has an amino acid sequence of SEQ ID NO:2.
In another embodiment the iENP is encoded by a nucleotide sequence having at least 80% similarity with the coding sequence of SEQ ID NO:7. In another embodiment the iENTP is encoded by a nucleotide sequence having at least 80% identity with the coding sequence of SEQ ID NO:7. In still another embodiment the IENT? has an amino acid sequence of SEQ ID NO:8 comprising one or more conservative substitutions thereof. In a preferred embodiment the isolated iENTP has an amino acid sequence of SEQ ID NO:8.
The present invention also includes modified iENTPs of the present invention, such as tagged proteins, labeled proteins, fusion proteins and the like. Such modified iENTPs may be used for example as antigens or for marker purposes. In a particular embodiment of this type, the fusion protein comprises an iENTP protein or active fragment thereof having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:2 comprising a conservative substitution thereof. In preferred embodiments the modified iENTP retains its activity as an NBMPR insensitive equilibrative nucleoside transport protein.
In a specific embodiment, an iENTP fusion protein can be expressed. An iENTP fusion protein comprises at least a functionally active portion of a non-iENTP protein joined via a peptide bond to at least a functionally active portion of an iENTP polypeptide. In a particular embodiment, an iENTP fusion protein or peptide contains an iENTP or fragment thereof and a FLAG-tag. In an alternative embodiment, an iENTP fusion protein or peptide contains an iENTP or fragment thereof and green fluorescent protein or derivatives thereof, as exemplified in U.S. Pat. No. 5,625,048 Issued Apr. 4, 1997 and International Publication No: WO 97/26333, hereby incorporated by reference in their entireties, can also be used.
The non-iENTP sequences of the iENTP fusion protein can be amino- or carboxy-terminal to the iENTP sequences. More preferably, for stable expression of an iENTP fusion protein (including a proteolytically inactive iENTP fusion protein), the portion of the non-iENTP fusion protein is joined via a peptide bond to the amino terminus of the iENTP protein. A recombinant DNA molecule encoding such a fusion protein comprises a sequence encoding at least a functionally active portion of a non-iENTP protein joined in-frame to the iENTP coding sequence. In one such embodiment the DNA molecule encodes a cleavage site for a specific protease, e.g., thrombin or Factor Xa, preferably at the iENTP-non-iENTP juncture. In a specific embodiment, the fusion protein is expressed in Escherichia coli. 
Antibodies to the iENTPs of the present invention are also part of the present invention. In a particular embodiment the antibody is raised against an iENTP having an amino acid sequence of SEQ ID NO:2. In another such embodiment the antibody is raised against an iENTP having an amino acid sequence of SEQ ID NO:2 comprising one or more conservative substitutions thereof. In still another embodiment the antibody is raised against a portion of, or alternatively all of the N-terminal 92 amino acids of SEQ ID NO:2, i.e., amino acids 1-92 of SEQ ID NO:2.
In one embodiment the antibody is a polyclonal antibody. In another embodiment the antibody is a monoclonal antibody. In yet another embodiment the monoclonal antibody is a chimeric antibody. The present invention also includes an immortal cell line that produces a monoclonal antibody of the present invention.
Still another aspect of the present invention includes a transfected or transduced cell in which all detectable nucleoside transport activity is performed by the nucleoside transport protein encoded by a nucleic acid of the present invention. In one embodiment of this type, the transfected or transduced cell is a vertebrate cell. In a preferred embodiment the transfected or transduced cell is a mammalian cell. In a more preferred embodiment the transfected or transduced cell is a human cell. In one such embodiment, the transfected cell is a human T-cell leukemia CEM cell. In a more particular embodiment of this type the transfected human @cell is a CEM/N1-7 cell. In a preferred embodiment of this aspect of the present invention, all detectable nucleoside transport activity is performed by an iENTP having the amino acid sequence of SEQ ID NO:2, or an active fragment of that iENTP. In a related embodiment the iENTP has the amino acid sequence of SEQ ID NO:2 comprising a conservative substitution thereof, or an active fragment of that iENTP.
The present invention also includes a nucleoside transport deficient subline of a human T-cell leukemia cell line CEM, transfected with an Epstein-Barr Nuclear Antigen 1 expression cassette, in which the cell line is capable of supporting the episomal replication of an Epstein-Barr virus-based mammalian expression vector. In one particular embodiment of 15 this type the expression vector is pDR2. In a preferred embodiment of this type the cell line has a stable transfection frequency with pDR2 of approximately 10xe2x88x922. In one particular embodiment the nucleoside transport deficient subline is CEM/C19.
Ribozymes specifically designed to modify the nucleic acids of the present invention are also contemplated as part of the present invention. Similarly antisense nucleic acids that hybridize under physiological conditions to an MRNA encoding an iENTP of the present invention is also included in the present invention. In one such embodiment, the antisense nucleic acid hybridizes to the mRNA that corresponds to the sense strand of nucleotides 238-1605 of the nucleotide sequence of SEQ ID NO:1.
A related aspect of the invention is a knockout mouse for the iENTPs of the present invention. One such embodiment comprises a first and a second allele which naturally encode and express the nucleoside transport protein having the amino acid sequence of SEQ ID NO:2. Both the first allele and the second allele each contain a defect which prevents the knockout mouse from expressing a nucleoside transport protein that is both insensitive to NBMPR and can function as an equilibrative nucleoside transport protein. Such a knockout mouse is particularly susceptible to drugs such as NBMPR.
The present invention also includes methods of making and using the iENTPs, antibodies to the iENTPs, the nucleic acids encoding the iENTPs, oligonucleotides that hybridize to these nucleic acids, DNA constructs containing these nucleic acids, cells containing these constructs, as well as to the other compositions and processes of the present invention.
Accordingly, one aspect of the present invention includes a method of isolating a cDNA encoding a nucleoside transport protein. This process comprises transfecting a nucleoside transport protein deficient cell with an expression vector from an expression vector library, wherein the expression vector library contains a vector comprising a cDNA encoding a nucleoside transport protein. The cDNA encoding the nucleoside transport protein is expressed in the transfected cell. An expression vector containing the cDNA encoding a nucleoside transport protein is selected by culturing the transfected cell under conditions in which the cell growth is dependent on the expression of the nucleoside transport protein. Therefore the selected expression vector contains the cDNA encoding a nucleoside transport protein. The selected expression vector is extracted from the transfected cell. A host cell is transfected with the selected expression vector, and the cDNA encoding the nucleoside transport protein is isolated.
In a specific embodiment of this type includes a method of isolating a cDNA encoding an NBMPR insensitive, equilibrative nucleoside transport protein (iENTP cDNA). This process comprises transfecting a nucleoside transport protein deficient cell with an expression vector from an expression vector library, wherein a cDNA library containing an iENTP cDNA has been subcloned into the expression vector library, and wherein the iENTP cDNA is expressed in the transfected cell. An expression vector containing the iENTP cDNA is selected by culturing the transfected cell under conditions in which the cell growth is dependent on the expression of the iENTP and its corresponding transport activity, and wherein the selected expression vector contains the iENTP cDNA. The selected expression vector is extracted from the transfected cell. A host cell is transfected with the selected expression vector, and the cDNA encoding the NBMPR insensitive, equilibrative nucleoside transport protein is isolated. In a preferred embodiment of this type the transfected cell is a human cell that expresses EBNA-1 and the human cell is CEM/C19.
Another aspect of the present invention includes a method of making an NBMPR insensitive, equilibrative nucleoside transport protein of the present invention through introducing an expression vector comprising a nucleic acid encoding the iENTP or an active fragment thereof into a host cell, and expressing the nucleic acid in the host cell. In one embodiment the host cell is a prokaryotic cell. In another embodiment the host cell is a eukaryotic cell. In one specific embodiment, the eukaryotic cell is an insect cell. In a particular embodiment the iENTP has an amino acid sequence of SEQ ID NO:2. In another particular embodiment the iENTP has an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution thereof. In one embodiment, the method further comprises purifying the iENTP.
The present invention includes methods for obtaining a purified NBMPR insensitive, equilibrative nucleoside transport protein (iENTP) or an active fragment thereof, from a cell that expresses the iENTP which comprises lysing the cell, and purifying the NBMPR insensitive, equilibrative nucleoside transport protein. In one embodiment the purifying step includes extracting the iENTP from the plasma membrane of the cell. In another such embodiment the purifying step also includes fractionating the proteins contained in the cell. In a particular embodiment, the iENTP is obtained from a natural source. In a preferred embodiment the natural source is a mammalian cell. In another particular embodiment the iENTP is a recombinant protein obtained from a prokaryotic cell. In still another embodiment the iENTP is a recombinant protein obtained from a eukaryotic cell. In one preferred embodiment the iENTP has an amino acid sequence of SEQ ID NO:2. In another preferred embodiment the iENTP has an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution thereof.
Yet another aspect of the invention includes a method of identifying a ligand of an iENTP of the present invention which comprises contacting a potential ligand with the isolated iENTP under physiological conditions (e.g., neutral pH, buffered solution with approximately 150 mM salt) and detecting whether the potential ligand binds to the iENTP wherein a potential ligand is selected as a ligand if it binds to the iENTP. The ligand and/or the iENTP can be labeled such as with a label defined below. Similarly, either the iENTP or ligand can be attached to a solid support. The binding can be detected with any of the standard protein-ligand binding assays known in the art as exemplified below. Once a ligand is identified its dissociation constant can be determined. Alternatively, the detecting step may be performed by determining the dissociation constant initially. In either case a potential ligand is selected as a ligand when the dissociation constant is less than 10xe2x88x92M. In one such embodiment the ligand is a permeant of the iENTP. In another embodiment, the ligand is an inhibitor of the iENTP. In yet another embodiment, the ligand is both a permeant and an inhibitor of the iENTP. In one preferred embodiment the iENTP has an amino acid sequence of SEQ ID NO:2. In another preferred embodiment the iENTP has an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution thereof.
The present invention also includes specific methods of identifying a permeant of an NBMPR insensitive, equilibrative nucleoside transport protein (iENTP). In one such embodiment a nucleoside or nucleoside analog is contacted with a transfected or transduced cell of the present invention in which all detectable nucleoside transport activity is performed by an iENTP of the present invention. The nucleoside transport of the nucleoside or nucleoside analog by the transfected or transduced cell is evaluated, wherein the nucleoside or nucleoside analog is identified as a permeant when the transport of the nucleoside or nucleoside analog into the transfected or transduced cell is determined to follow a facilitated diffusion process. In one such embodiment the nucleoside or nucleoside analog is an antiviral nucleoside analog. In another embodiment the nucleoside or nucleoside analog is an antitumor nucleoside analog. In one particular embodiment of this type the transfected or transduced cell is a transfected or transduced human cell. In one preferred embodiment the iENTP has an amino acid sequence of SEQ ID NO:2. In another preferred embodiment the iENTP has an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution thereof.
The present invention further includes specific methods of selecting drugs that inhibit an NBMPR insensitive, equilibrative nucleoside transport protein. One such embodiment comprises contacting a potential drug with a transfected or transduced cell of the present invention in which all detectable nucleoside transport activity is performed by an iENTP of the present invention. The nucleoside transport activity of the cell is evaluated. A potential drug is selected as a drug when a decrease in the nucleoside transport activity is determined relative to that determined when the evaluating was performed in the absence of the potential drug.
In one embodiment of this type the nucleoside transport activity of the transfected or transduced cell is evaluated as a function of the determination of the trans-stimulation of a permeant. In another embodiment the nucleoside transport activity of the transfected or transduce cell is evaluated as a function of the determination of the direct transport of a permeant. In still another embodiment the nucleoside transport activity of the transfected or transduced cell is evaluated as a function of the determination of the countertransport of a permeant. In one specific embodiment, the nucleoside transport activity of the transfected or transduced cell is evaluated as a function of the toxicity of a nucleoside analog which is a permeant of the iENTP, such as tubercidin, 2-chloro-2xe2x80x2-deoxyadenosine, or Ara-C. In yet another embodiment, the nucleoside transport activity of the transfected or transduced cell is evaluated as a function of toxicity in the presence of an antimetabolite. In yet another embodiment the nucleoside transport activity of the transfected or transduced cell is evaluated as a function of two of these determinations. In still another embodiment the nucleoside transport activity of the transfected or transduced cell is evaluated as a fuinction of all of these determinations.
Another embodiment of a method of selecting a drug that inhibits an NBMPR insensitive, equilibrative nucleoside transport protein (iENTP) comprises detecting the mutual inhibition (i.e. mutual competition) of a potential drug with a perrneant, such as uridine for the iENTP in a transfected or transduced cell of the present invention in which all detectable nucleoside transport activity is performed by an iENTP of the present invention. A potential drug is selected as a drug when mutual inhibition is detected. This embodiment may be used alone or in conjunction with the other determinations described above.
For any of the drug assays of the present invention the iENTP. functions as an equilibrative nucleoside transport protein, is insensitive to NBMPR, and contains approximately 450 amino acid residues. In one particular embodiment the iENTP has an amino acid sequence of SEQ ID NO:2. In another embodiment the iENTP has an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution thereof. In a preferred embodiment the transfected or transduced cell is a human transfected or transduced cell.
Accordingly, it is a principal object of the present invention to provide a purified NBMPR-insensitive, equilibrative nucleoside transport protein (iENTP).
It is a further object of the present invention to provide an isolated nucleic acid encoding a iENTP.
It is a further object of the present invention to provide a DNA construct containing a nucleic acid encoding a iENTP.
It is a further object of the present invention to provide an antibody specific for a purified iENTP.
It is a further object of the present invention to provide a method of producing an iENTP, including through modification of a iENTP, and through recombinant technology.
It is a further object of the present invention to provide a method of selecting a drug that preferentially inhibits an iENTP-dependent nucleoside transport pathway.
It is a further object of the present invention to provide a method of screening drug libraries for drugs that preferentially inhibit an iENTP.
It is a further object of the present invention to provide a cell in which the only detectable facilitated diffusion pathway for nucleosides includes an iENTP.
It is a further object of the present invention to provide a cell where the NBMPR-insensitive facilitated diffusion pathway for nucleosides includes a modified iENTP.
It is a further object of the present invention to provide a method of cancer chemotherapy by transducing hematopoietic stem cells ex vivo with a cDNA encoding an iENTP, introducing the transduced cells into an animal subject, and then treating the animal subject with an antimetabolite and NBMPR.
It is a further object of the present invention to provide a novel method of hematopoietic cell-directed gene therapy using an expression vector encoding the iENTP.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.